The absence of a suitably HLA-matched donor precludes hematopoietic cell transplantation (HCT) in many patients, as the risks of graft-versus-host disease (GVHD) and rejection are formidable when extensive HLA barriers are transgressed. HLA-mismatched HCT without rejection or severe GVHD would make this treatment available to patients lacking a matched donor, and could maximize graft-versus-tumor (GVT) effects. The toxicity of allogeneic HCT can be diminished using non-myeloablative conditioning. The major goal of this Program Project is to develop non-myeloablative approaches to HLA-mismatched HCT, while minimizing GVHD and exploiting the potent GVT effect of anti-MHC GVH alloreactivity. Two major strategies will be explored in animal models, and a third will be characterized mechanistically. Nonmyeloablative allogeneic HCT that includes T cell depletion of the recipient and donor inoculum can lead to mixed hematopoietic chimerism across extensive MHC barriers without GVHD. When donor lymphocyte infusions (DLI) are administered to established murine mixed chimeras, the GVH alloresponse associated with subsequent DLI leads to powerful GVT effects without GVHD. Recipient-derived professional antigenpresenting cells (APC) in mixed chimeras maximize GVH alloresponses and thereby maximize GVT, while GVHD, a disease of epithelial target tissues, is avoided. We have taken these observations from mice to a unique large animal (pig) model and into clinical trials for the treatment of leukemias and lymphomas. Two of the Projects (Project 1 in mice, Project 3 in large animals) focus on the administration of delayed DLI to mixed allogeneic chimeras prepared with non-myeloablative conditioning that includes T cell-depleting mAbs, in order to achieve maximal GVL effects without GVHD. The goals of expanding donor hematopoietic stem cells (HSC) ex vivo and in vivo and improving immune recovery in Project 4 will ultimately be applicable to the clinical application of this approach, since large numbers of purified (T cell-depleted) donor HSC may be required to achieve engraftment across extensive HLA barriers. Project 4 includes an initial clinical evaluation of the ability of PTH to expand the numbers of mobilized HSC in patients failing conventional mobilization treatment. Large animal studies to evaluate the use of PTH to enhance allogeneic HSC engraftment (Project 3) will provide a critical bridge between the pre-clinical and clinical applications of these strategies (Project 4). Like Project 1, Project 2 involves confinement of the GVH alloresponse to the lymphohematpoietic system, via a novel strategy involving IFN-gamma. A mechanistic understanding of this phenomenon will lead to the development of novel strategies for separating GVHD and GVL clinically. Core A will provide multicolor (up to 11-color) flow cytometry and high speed cell sorting services to support all of these projects. Core B (Administrative) will be responsible for administering the grant, and Core C will provide statistical and database support for all of the Projects.